During the past year, we have been focusing on two areas of research. 1. Study structural biology of VEGF 3-prime UTR RNA and its interaction with protein regulators; 2. Study the structure basis of the adenine riboswitch; 3. Develop tools and methods that facilitate the studies as well as have a significant impact to scientific communities and potential applications for imaging and disease detection.1. The gene expression of VEGF is regulated at multiple stages, including but not limited to post-transcription and translational levels. The down-regulation of the VEGF expression has been mapped to specific interactions between RNA125 and one of proteins in the GAIT complex. An earlier study by Dr. Fox's laboratory showed that the down-regulation involves specific interactions between the linker domain of the glutamyl-prolyl tRNA synthetase (EPRS), which is one of components of the GAIT complex, and a 29-nt hairpin (H29) that is entirely composed of A or U nucleotides and is the part of the 125-nt RNA . The structural basis for this important interaction is unknown. The EPRS linker domain (ELD) consists of three dual-helical bundles and the interaction site for H29 has been mapped to the first two helical bundles, r1r2, by Dr. Fox's group using biochemical methods. r1r2 contains 114 amino acids and the three-dimensional structure of the first dual-helical bundle has been reported. We are currently investigating the structural basis of down- and up-regulation of the VEGF expression using combination of NMR and SAXS in a collaboration with Professor Paul Fox's group of the Cleveland Clinic. We have encountered a significant difficult to obtain highly pure RNA samples suitable for NMR study. Nevertheless, we have determined three-dimensional structures of r1r2 and the H29 RNA, and we are in the process to determine the complex structure between r1r2 and H29.2. We have obtained samples of the adenine riboswitch and its mutants in large quantity and performed preliminary NMR experiments. However, we have to develop new methods for structure determination of this RNA. One of these new methods is described in the following section.3. My group has developed a new method for selective labeling of RNA (SLOR) at designated residue(s) and/or segment(s) of large RNAs using solid-phase multi-cycle enzymatic reactions. The potential applications of SLOR are broad and far reaching, due to a wide range of roles that RNA plays in biology. The followings are just examples of a few areas.General RNA biochemistry, biophysics and molecular biology. The fluorescent labeled RNA molecules can be used to study interaction between/within RNAs, and between RNA and DNA, RNA and proteins in vitro or within the cellular environment following microinjection. For example, selectively labeled RNA can be used to study riboswitch mechanisms in regulation of gene expression. The fluorescent residues can be incorporated at two strategically locations in the riboswitch using SLOR in order to monitor the switching event that is directly synchronized with the relative movement between the aptamer and expression platform domains using time-resolved single molecule Forster Resonance Energy Transfer (FRET) experiments. Probing such an event has not been possible because of lack of the specifically fluorescent labeled riboswitch RNA molecules. RNA structural biology. RNA molecules alone are almost impossible to crystallize for structure determination. NMR spectroscopy is an ideal method for structure determination of RNAs since it is a solution-state method and does not require crystallization. However, it is limited to only small RNAs, up to 50 residues, because of the extensive overlaps of chemical shift signals and short lifetimes of NMR signals. With SLOR, selectively labeled RNAs at designated residue(s) and/or segment(s) can be used for recording NMR signals, resulting in greatly simplified NMR spectra for straightforward interpretation. Moreover, one can selectively deuterate designated residues/segments and record the signals from the remaining residues. The signals from the remaining residues will have a much longer lifetime, resulting in significant enhancement of both resolution and sensitivity of NMR signals. This enhancement will make it possible to determine high-resolution structures of much larger RNAs using NMR spectroscopy: this will revolutionize RNA structural biology.The SLOR method will have an immediate impact to several collaborations between my group and several other groups within NCI (those collaborations are part of reasons that I developed SLOR). For example, we are collaborating with Dr. Alan Reins group studying the dimerization of the genomic RNAs of HIV-1 and murine leukemia virus. Dimerization of these RNAs is critical for viral packaging and maturation. The minimum size of the viral RNA that behaves like the whole viral genomic RNA is ca. 170 nucleotides (nt). Using the SLOR method, we can place fluorescent residues at the selected locations to monitor the dimerization and viral packaging using FRET and imaging experiments. Moreover, we can determine the three-dimensional (3D) structure of the RNA dimer by recording NMR spectra of the selectively isotope-labeled RNA samples. Another example is our collaboration with Dr. LeGrices group to determine the 3D structure of the HIV Rev Response Element (RRE), a 267-nt RNA, and to study the interaction of RRE with the Rev protein. 3D structure determinations of both the RNAs (for encapsidation and for Rev recognition) have not been feasible because of their sizes and the limitations of NMR and X-ray crystallography. Moreover, in principle, the SLOR method may be useful in the research of any NCI intramural investigators who study RNA biology or use RNA-based probes for detection, imaging and therapeutic applications.Clinic application. The discovery of disease-causing mutations in RNAs is yielding a wealth of new therapeutic targets, and the growing understanding of RNA biology and chemistry provides new RNA-based tools for developing therapeutics. There is a boom in applications and development of RNA-based reagents for detection, diagnosis and therapy in the past two decades. Among those RNA-based reagents, RNA aptamers are particularly useful because of their wide range of application against a variety of targets, including but not limited to organic dyes and amino acids, antibiotics, peptides, proteins of various sizes, functions, whole cells, viruses as well as specific molecular markers in various cancers. Potentially, the labeled RNAs or RNA aptamers produced using SLOR can be used to identify or improve these interactions at a resolution of individual residues.Patent applications. The SLOR method and the SLOR synthesizer may have wide potential commercial applications and may be filed for two separate patent applications. Companies that provide DNA and/or RNA and/or peptide synthesis would be potential licensees for licensing the SLOR method; companies that specialized in RNA synthesis or manufacture DNA or peptide synthesizers may be potential licensees of the SLOR RNA synthesizer. The number of those companies ranges at least several tens in US alone.